A majority of human cancers arise in epithelial tissue. In the body, epithelial cells separate the internal living stromal tissue from the exterior environment. Epithelial cells exist either as stratified layers that directly face the exterior environment (such as the epidermis) or as a monolayer that can be folded into circular acini or ducts containing lumina.
An epithelial cell is functionally polarized with respect to the two environments it separates. The basal surface of the epithelial is connected to extracellular matrix material which is organized as a basal lamina adjacent to the extracellular matrix of the stromal. The apical surface is free of an apposed extracellular matrix layer, and serves as a boundary to molecular diffusion.
In mammary tissue, the polarity of normal epithelial cells apparently is directed by attachment of the cells to the basal surface. Tumor cells seem to lose this polarity, however. Metastatic epithelial tumor cells secrete hydrolytic enzymes that degrade the basal lamina before they invade the stroma of the breast. Those tumor cells that have degraded the basal lamina lose their polarity, while those cells that remain in contact with non-degraded basal lamina remain polarized.
This difference in morphology between normal and tumor epithelial cells in vivo cannot be reproduced in vitro for several reasons. First, it is very difficult to grow normal epithelial cells in culture at all. Second, because the normal cells grown in culture do not reproduce epithelial morphology observed in vivo, it is difficult to distinguish between normal and transformed cells in culture, although such distinctions are crucial to investigating early events in tumor development. For example, investigations of breast cancer have relied upon the difference in longevity between normal and transformed cells in culture; this difference is the senescence of normal cells and the immortal growth of tumor cells. These in vitro phenomena take time, however, and do not have clear counterparts in vivo.
It would be most useful if epithelium tissue could be grown in vitro conveniently and would exhibit the morphological, physiological, and biochemical behavior characteristic of epithelial cells grown in vivo. These capabilities would allow investigation in vitro of both the development of epithelial cancer, in terms of what triggers such a cancer and how it proceeds, and how better to treat or prevent it.
Because it is difficult to grow normal epithelial cells in culture, most cancer researchers utilize immortal cell lines which are derived from other sources. For example, most studies of breast cancer biology have relied upon a few cell lines which are primarily derived from human breast cancer metastases or rodent primary cells. The best known lines (for example, MCF-7 and MDA-MB231, see Table 1) were generated from metastasized, drug resistant tumors; the lines were established from samples taken from patients who had already undergone radiation therapy and chemotherapy. These tumor lines represent the extreme end of the tumor progression spectrum, and are therefore limited in their usefulness as research tools. In addition to the lack of early stage tumor lines, only one "normal" breast cell line is available (MCF10, see Table 1), and it is not derived from epithelial cells.
TABLE 1 ______________________________________ Origin of frequently studied breast tumor cell lines Cell Line Origin (Source) ______________________________________ MDA-MB231 Pleural effusion from a 51 yr old Caucasian female with adenocarcinoma of the breast treated with 5-fluorouracil, prednisone, cytoxan, adriamycin, and methotrexate (ATCC, passage 14) BT20 Infiltrating ductal carcinoma of the breast from a 74 year old Caucasian female (American Type Tissue Culture, ATCC, passage 248) MCF7 Pleural effusion from a postmenopausal woman with adenocarcinoma of the breast treated with radiation and hormones (ATCC, passage 138) MC7/LY2 Pleural effusion cells resistant against LY117018 (an estrogenic drug) (ATCC) CAMA-1 Malignant pleural effusion of a postmenopausal woman with adenocarcinoma of the breast (ATCC, passage 21) SKBR# Malignant pleural effusion from a 43 year old Caucasian female with adenocarcinoma of the breast treated with radiation, steroids, cytoxan, and 5-fluorouracil MCF10A or F Myoepithelial cells immortalized with an exogenous agent and derived from normal breast reduction tissue (Michigan Cancer Foundation) ______________________________________
Primary human normal and tumor-derived breast epithelial cells have only recently been cultured. While both types of cells are difficult to culture, breast carcinomas remain virtually intractable. For example, MDA-MB231 was derived from the 231st attempt to generate a cell line by placing human tumor cells into an immunodeficient mouse strain.
Those cell lines which are available suffer from several disadvantages. The lines represent highly invasive tumor cell types, since most are derived from pleural effusions. They do not represent different stages of tumor growth, as most represent late stage tumors, and they do not represent non-invasive tumor types such as Ductal Carcinoma In Situ ("DCIS"). Furthermore, the lines have undergone many passages. Tumor cells are known for their genomic instability and may further diverge genetically even after establishment of a cell line. Thus, cell lines many generations removed from the original tumors may have undergone selective pressures and clonal evolution. This process can create cell lines that bear little genetic, biochemical, and morphological resemblance to the original tumor from which they are derived. Finally, very little information is available about the original sources of the previously available lines.
Human mammary epithelial cells are typically grown in monolayer culture on tissue culture plastic surfaces in medium containing low concentrations of either serum or pituitary extract. Attempts to grow these cells in a more physiological manner have involved coating the plastic culture surface with either rat tail collagen or a commercially available matrix, MATRIGEL.RTM. Matrix.
Conventional methodology for culturing human mammary epithelial involves three steps. The first is to isolate epithelial cells from the tissue sample, primarily by the use of digestive enzymes, resulting in free cells and cell clumps, which can be separated from non-epithelial material. The second step is to place the cells on a surface, which is typically plastic. The third step is to surround the cells with a medium, which typically contains minimal essential nutrients, such as sugars and amino acids, as well as serum (Band, V. and Sager, R (1989) Proc. Nat'l Acad Sci (USA) 86: 1249-1253) or pituitary extract (Hammond, SL, et al. (1984) Proc Nat'l Acad Sci (USA) 81: 5435-5439).
A recent improvement includes coating the culture surface with a material to mimic the extracellular matrix in vivo. The material currently used is rat tail collagen (Yang, J, et al. (1980) J Nat'l Cancer Inst 65: 337-343; Hall, HG, et al. (1982) Proc Nat'l Acad Sci USA 79: 4672-4676) or MATRIGEL.RTM. Matrix. Recent reports (Peterson, OW, et al. (1992) Proc Nat'l Acad Sci (USA) 89: 9064-9068; Bergstraesser, LM and Weitzman, SA (1993) Cancer Research 53: 2644-2654) indicate that normal human epithelium, grown on MATRIGEL.RTM. Matrix in a complex culture medium which included hormones and growth factors, formed three-dimensional luminal structures called "organoids," while malignant cells remained as single cells and migrated through the MATRIGEL.RTM. Matrix. The "organoids" appeared as large structures made of hundreds of cells and many cell layers thick. On the other hand, this system did not form duct-like structures or other structures of increasing complexity. Also, the cultures were not long-lived, surviving for only seven days. These culture methods thus are of limited value for investigating normal epithelial tissue growth and the early events of epithelial tumor development. Such investigations appear to require the existence of viable epithelial stem cells which have the potential to differentiate in vitro into the complex architecture that is normally observed in vivo.